Visualization of clonal expansion after massive depletion of cells carrying the bovine leukemia virus (BLV) integration sites during the course of disease progression in a BLV naturally-infected cow: a case report

Saito, Susumu; Hosomichi, Kazuyoshi; Yamanaka, Meripet Polat; Mizutani, Tetsuya; Takeshima, Shin‐nosuke; Aida, Yoko

doi:10.1186/s12977-022-00609-0

Cited by 3 publications

(1 citation statement)

References 40 publications

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“…We do not determine the integration site of TERT in the bat chromosome in this study. According to the retrovirus studies [ 41 , 42 , 43 ], the retrovirus genes integrate randomly into the host cell DNA genome at an early stage and are removed/deleted by the DNA repair function and immunity. As a result, the number of polyclonal integration sites decreases.…”

Section: Discussionmentioning

confidence: 99%

Establishment of immortalized Egyptian Rousettus bat cell lines

Bai,

Tani,

Kobayashi

et al. 2024

FEBS Open Bio

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The Egyptian Rousettus bat (Rousettus aegyptiacus) is a common fruit bat species that is distributed mainly in Africa and the Middle East. Bats serve as reservoir hosts for numerous pathogens. Human activities, such as hunting bats for food, managing vermin, and causing habitat loss, elevate the likelihood of transmission of bat pathogens to humans and other animals. Consequently, bat cell lines play a crucial role as research materials for investigating viral pathogens. However, the inherent limitation of finite cell division in primary cells necessitates the use of immortalized cells derived from various bat tissues. Herein, we successfully established six fibroblast cell lines derived from an infant bat heart and lungs and an elderly bat heart. Three of the six cell lines, called K4DT cells, were transduced by a combination of cell cycle regulators, mutant cyclin‐dependent kinase 4, cyclin D1, and human telomerase reverse transcriptase. The other three cell lines, named SV40 cells, were transfected with simian virus 40 large T antigen. Transgene protein expression was detected in the transduced cells. All three K4DT cell lines and one lung‐derived SV40 cell line were virtually immortalized and nearly maintained the normal diploid karyotypes. However, the two other heart‐derived SV40 cell lines had aberrant karyotypes and the young bat‐derived cell line stopped proliferating at approximately 40 population doublings. These bat cell lines are valuable for studying pathogen genomics and biology.

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Section: Discussionmentioning

confidence: 99%

Establishment of immortalized Egyptian Rousettus bat cell lines

Bai,

Tani,

Kobayashi

et al. 2024

FEBS Open Bio

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The influence of risk factors on bovine leukemia virus infection and proviral load in egyptian cattle

Hamada,

Fereig,

Metwally

2023

Vet Res Commun

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A rapid and simple clonality assay for bovine leukemia virus-infected cells by amplified fragment length polymorphism (AFLP) analysis

Kobayashi,

Makimoto,

Ohnuki

et al. 2024

Microbiol Spectr

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Enzootic bovine leukosis (EBL), although eradicated in some European countries, is still the most common neoplastic disease of cattle, caused by the bovine leukemia virus (BLV). During the progression of EBL, BLV-infected cells clonally expand, and some of which result in tumor onset. The clonality of BLV-infected cells is generally evaluated with NGS or Sanger sequencing. Although these methods clearly distinguish EBL from non-EBL cases, the procedures are complex and not practical for routine veterinary diagnosis. In this study, we developed an amplified fragment length polymorphism (AFLP) analysis for BLV clonality assay (BLV-AFLP). This analysis uses restriction enzyme digestion to amplify the chimeric regions of BLV 3’ linear transcribed region (LTR) and host genome through conventional polymerase chain reaction (PCR) and visualizes the results by gel-electrophoresis. The method was established using cattle samples representing different stages of the disease: BLV-uninfected, non-EBL, and EBL cattle. Non-EBL cattle showed smeared bands, indicating polyclonal proliferation, while EBL cattle showed distinct bands, indicating clonal expansion. The results of BLV-AFLP correlated well with those of previously reported methods, suggesting its efficacy in detecting clonal proliferation. The validation using blood samples of non-EBL cattle and tumor samples of EBL cattle confirmed that BLV-AFLP could effectively identify clonal proliferation in EBL samples. Moreover, the emergence of dominant clones in the tumor at later stages was successfully detected before EBL onset in some cattle, highlighting its sensitivity and potential for early detection. Overall, BLV-AFLP is suitable for practical use in the field, improving BLV management strategies and minimizing economic losses. IMPORTANCE Enzootic bovine leukosis (EBL) is routinely diagnosed based on external manifestations at the farm, such as the presence of tumors and/or general lymph node enlargement. However, due to the nonspecific clinical manifestations of EBL, over half of EBL cases are unrecognized at the farm, with most cases being diagnosed during postmortem inspection at the slaughterhouse. Early detection and monitoring of clonal expansion are necessary for managing EBL and reducing economic losses. In this study, we developed BLV-AFLP that represents a significant advancement in the diagnosis of EBL in cattle. This method can rapidly assess the clonal proliferation of BLV-infected cells, crucial for distinguishing between asymptomatic and EBL cattle. Additionally, tracking clonal dynamics offers insights into the disease’s progression, potentially providing strategies for avoiding economic losses. Overall, as BLV-AFLP is a simple and rapid test for detecting EBL, it is feasible and efficient for routine veterinary practice.

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Visualization of clonal expansion after massive depletion of cells carrying the bovine leukemia virus (BLV) integration sites during the course of disease progression in a BLV naturally-infected cow: a case report

Cited by 3 publications

References 40 publications

Establishment of immortalized Egyptian Rousettus bat cell lines

Establishment of immortalized Egyptian Rousettus bat cell lines

The influence of risk factors on bovine leukemia virus infection and proviral load in egyptian cattle

A rapid and simple clonality assay for bovine leukemia virus-infected cells by amplified fragment length polymorphism (AFLP) analysis

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